Estrogen related receptor, ERRalpha, a regulator of bone formation

ABSTRACT

Methods and pharmaceutical compositions are provided for modulating bone formation in a mammal. Methods are also provided for screening compounds for their efficacy as modulators of bone formation.

RELATED APPLICATION INFORMATION

This application is a continuation of co-pending U.S. application Ser.No. 10/089,429, filed Nov. 29, 2002, which claims the benefit under 35U.S.C. §371 from PCT Application No. PCT/CA00/01015, filed Aug. 30,2000, which claims the benefit of Canadian Application Serial No.2,284,103, filed Sep. 30, 1999, the disclosures of which areincorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to methods and pharmaceutical compositionsfor modulation of bone formation.

BACKGROUND OF THE INVENTION

In the description which follows, references are made to certainliterature citations which are listed at the end of the specificationand all of which are incorporated herein by reference.

Nuclear receptors are transcription factors involved in variousphysiological regulatory processes. The superfamily to which nuclearreceptors belong comprises both ligand-dependent molecules such as thesteroid hormone-, thyroid hormone-, retinoic acid- and vitaminD-receptors, and an increasing number of so-called orphan receptors forwhich no ligand has yet been determined (Gronemeyer H, Laudet V., 1995;Enmark and Gustafsson,1996). Indeed, it is not yet known whether theorphan receptors have ligands that await identification or whether theyact in a constitutive manner. The orphan receptors display the samestructural organization as do the classic ligand-dependent receptors:the A/B domain located in the N-terminal part of the protein harbors aligand-independent transactivation function (AF-1); the C domain, whichis the most conserved part of the molecule, is responsible for thespecific DNA-binding activity; the E domain contains the ligand bindinghydrophobic pocket and contributes to receptor dimerization and to theligand-dependent transactivation function (AF-2).

Two orphan receptors, estrogen receptor-related receptor α (ERRα) andERRβ (Giguere et al., 1988; NR3B1 and NR3B2, respectively, according tothe Nuclear Receptors Nomenclature Committee, 1999) are closely relatedto the estrogen receptors ERα and ERβ (Green et al., 1986; Kuiper etal., 1996; NR3A1 and NR3A2 respectively). ERRα (Genbank Accession No.for human ERRα: NM_(—)004451) and ERRβ were identified by low-stringencyscreening of cDNA libraries with a probe encompassing the DNA-bindingdomain of the human estrogen receptor (ER). Recently, a third estrogenreceptor-related receptor, ERR3 or ERRγ, was identified by yeasttwo-hybrid screening with the glucocorticoid receptor interactingprotein 1 (GRIP1) as bait (Hong et al, 1999). The DNA binding domainregion of ERRs and ERs is highly conserved, however the others parts ofthe protein share very little homology (Giguere et al, 1988; Hong et al,1999). Therefore, sequence alignment of ERRα and the ERs reveals a highsimilarity (68%) in the 66 amino acids of the DNA-binding domain and amoderate similarity (36%) in the ligand-binding E domain, which mayexplain the fact that ERRα does not bind estrogen. Although ligands forthe ERRs have not been clearly identified, the pesticides chlordane andtoxaphene have been suggested to be potential ligands for ERRα (Yang andChen, 1999). ERRα has been identified as a regulator of the SV40 majorlate promoter during the early-to-late switch of expression (Wiley etal., 1993) and as a regulator of fat metabolism (Sladek et al., 1997;Vega et al, 1997). Yang et al. also showed that ERR mo d ulates theactivating effect of estrogens on the lactoferrin promoter and suggestedthat ERRα may interact with ERs through protein-protein interaction(Yang et al., 1996; Zhang and Teng, 2000). Finally, ERRα has beendescribed as a modulator of the human aromatase gene in breast, andhypothesized to be critical for normal breast development and to play animportant role in the pathogenesis and maintenance of breast cancer viaits ability to interact with ERs (Yang et al, 1998).

Postmenopausal osteoporosis is a condition caused primarily by thesevere decrease of serum estrogen levels after cessation of ovarianfunction. The absence of estrogen results in an increase in boneturnover (Tumer et al, 1994) and a negative bone remodeling balance,leading to bone loss and an increased fracture risk. An anabolic effectof estrogens on bone homeostasis has been documented in post-menopausalosteoporosis (for review see Pacifi, 1996), where bone loss can bereversed by administration of natural or synthetic estrogens. Althoughthe bone preserving effect of estrogen replacement is indisputable, themolecular and cellular mechanism of action for this hormone effectremain unclear. ERs are expressed in osteoblasts (Turner et al., 1994;Eriksen et al, 1988; Komm et al, 1988), and estrogens have been found toelicit effects ranging from modulation of gene expression to regulationof proliferation in this cell type (for review Harris et al, 1996). Incontrast, mice lacking a functional ERα or ERβ have only minor skeletalabnormalities (Korach et al, 1994; Windahl et al, 1999) suggesting thatother mechanisms or receptors might be important during skeletaldevelopment. ERRβ expression is restricted to early development and to afew adult tissues (Giguere et al., 1988; Pettersson et al., 1996). Incontrast, ERRα has a broader spectrum of expression, including fat,muscle, brain, testis and skin (Bonnelye et al, 1997b). Strikingly, ERRαis also highly expressed in the ossification zones of the mouse embryo(in long bones, vertebrae, ribs and skull), and is more widelydistributed in osteoblast-like cells than is ERα (Bonnelye et al.,1997a). Morever it has been shown that ERRα positively regulates theosteopontin gene (Vanacker et al, 1998), an extracellular matrixmolecule secreted by osteoblasts and other cells and thought to play arole in bone remodelling among other functions (Denhardt and Noda,1998).

SUMMARY OF THE INVENTION

The inventors have demonstrated the involvement of ERRα in themodulation of bone formation in mammals. Up regulation of ERRα increasedosteoblast differentiation from progenitor cells and also proliferationof progenitor cells. Down regulation of ERRα caused inhibition of boneformation, with reduction of osteoblast numbers and differentiation.ERRα was shown to be expressed also in osteocytes in both calvaria andlong bones, indicating a role in skeletal maintenance.

In accordance with one embodiment of the present invention, a method ofincreasing proliferation of osteoblasts in a mammal comprisesadministering to the mammal an effective amount of an agent selectedfrom the group consisting of:

-   -   (a) an estrogen related receptor alpha (ERRα) agonist;    -   (b) a substantially purified ERRα protein; and    -   (c) a nucleotide sequence encoding ERRα protein.    -   (d) an agent which enhances expression of a gene encoding an        ERRα protein.

In accordance with another embodiment of the present invention, a methodof increasing differentiation of osteoblasts in a mammal comprisesadministering to the mammal an effective amount of an agent selectedfrom the group consisting of:

-   -   (a) an ERRα agonist;    -   (b) a substantially purified ERRα protein; and    -   (c) a nucleotide sequence encoding ERRα protein.    -   (d) an agent which enhances expression of a gene encoding an        ERRα protein.

In accordance with another embodiment of the present invention, a methodof reducing proliferation of osteoblasts in a mammal comprisesadministering to the mammal an effective amount of an agent selectedfrom the group consisting of:

-   -   (a) an ERRα antagonist;    -   (b) a purified antibody which binds specifically to an ERRα        protein;    -   (c) an antisense nucleotide sequence complementary to and        capable of hybridizing to a nucleotide sequence encoding an ERRα        protein; and    -   (d) an agent which reduces expression of a gene encoding an ERRα        protein.

In accordance with another embodiment of the present invention, a methodof reducing differentiation of osteoblasts in a mammal comprisesadministering to the mammal an effective amount of an agent selectedfrom the group consisting of:

-   -   (a) an ERRα antagonist;    -   (b) a purified antibody which binds specifically to an ERRα        protein;    -   (c) an antisense nucleotide sequence complementary to and        capable of hybridizing to a nucleotide sequence encoding an ERRα        protein; and    -   (d) an agent which reduces expression of a gene encoding an ERRα        protein.

In accordance with another embodiment of the present invention, a methodfor treating a disorder associated with bone loss in a mammal comprisesadministering to the mammal an effective amount of an agent selectedfrom the group consisting of:

-   -   (a) an ERRα agonist;    -   (b) a substantially purified ERRα protein; and    -   (c) a nucleotide sequence encoding ERRα protein.    -   (d) an agent which enhances expression of a gene encoding an        ERRα protein.

In accordance with another embodiment of the present invention, a methodfor treating a disorder associated with unwanted bone formationcomprises administering to the mammal an effective amount of an agentselected from the group consisting of:

-   -   (a) an ERRα antagonist;    -   (b) a purified antibody which binds specifically to an ERRα        protein;    -   (c) an antisense nucleotide sequence complementary to and        capable of hybridizing to a nucleotide sequence encoding an ERRα        protein; and    -   (d) an agent which reduces expression of a gene encoding an ERRα        protein.

In accordance with another embodiment of the present invention, a methodfor screening a candidate compound for its ability to modulate ERRαactivity comprises:

-   -   (a) providing a system for measuring a biological activity of        ERRα; and    -   (b) measuring the biological activity of ERRα in the presence or        absence of the candidate compound,        wherein a change in ERRα activity in the presence of the        compound relative to ERRα activity in the absence of the        compound indicates an ability to modulate ERRα activity.

In accordance with another embodiment of the present invention, a methodfor screening a candidate compound for potential efficacy in promotingbone formation comprises:

-   -   (a) providing an assay system for determining ERRα agonist        activity of a compound; and    -   (b) testing the candidate compound for ERRα agonist activity in        the assay wherein ERRα agonist activity in the candidate        compound indicates potential efficacy as a promoter of bone        formation.

In accordance with another embodiment of the present invention, a methodfor screening a candidate compound for potential efficacy in inhibitingbone formation comprises:

-   -   (a) providing an assay system for determining ERRα antagonist        activity of a compound; and    -   (b) testing the candidate compound for ERRα antagonist activity        in the assay wherein ERRα antagonist activity in the candidate        compound indicates potential efficacy as an inhibitor of bone        formation.

In accordance with another embodiment of the present invention, apharmaceutical composition comprises an effective amount of an agentselected from the group consisting of:

-   -   (a) an ERRα agonist;    -   (b) a substantially purified ERRα protein; and    -   (c) a nucleotide sequence encoding ERRα protein and a        pharmaceutically acceptable carrier.    -   (d) an agent which enhances expression of a gene encoding an        ERRα protein.

In accordance with another embodiment of the present invention, apharmaceutical composition comprises an effective amount of an agentselected from the group consisting of:

-   -   (a) an ERRα antagonist;    -   (b) a purified antibody which binds specifically to ERRα        protein;    -   (c) an antisense nucleotide sequence complementary to and        capable of hybridizing to a nucleotide sequence encoding ERRα        protein; and    -   (d) an agent which reduces expression of the gene encoding ERRα        protein        and a pharmaceutically acceptable carrier.

SUMMARY OF THE DRAWINGS

Certain embodiments of the invention are described, reference being madeto the accompanying drawings, wherein:

FIG. 1, Panel A is a Northern blot showing expression of ERRα, alkalinephosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN), in primaryrat calvaria (RC) cells over a proliferation-differentiation time coursein presence (+Dex) or absence (−Dex) of dexamethasone (Dex) duringproliferation (day 6), early nodule formation (day 10) and nodulemineralization (day 15).

FIG. 1, Panel B shows ERRα mRNA expression normalized against that ofthe ribosomal protein L32; the Y-axis is the ratio of the ERRα signal tothat of L32. For comparison, mRNA levels for three osteoblast markers,alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN), arealso shown (Panel A) and normalized against L32 (Panel B).

FIG. 2 shows detection of mERRα, (Type I collagen, osteopontin, alkalinephosphatase, bone sialoprotein and osteocalcin) by RT-PCR in librariesselected based on the basic molecular phenotype of the poly(A) PCRlibraries made from discrete isolated colonies at different stages ofosteoblast differentiation and bone development. Gene expressionprofiles of colonies were determined by analyzing expression of severalknown osteoblast lineage markers. These 19 libraries, from a pool of>100characterized libraries, were selected for fingerprinting on the basisthat they represent several transitional stages: primitive progenitors(A), progressively more mature precursors (B,C,D) and terminallydifferentiated, bone forming osteoblasts (E). While category order isprogressive, the order of colonies within each category is random.

FIG. 3 shows in Panel A: a Western blot of whole-cell extracts obtainedfrom Hela cells and, in Panel B: MC3T3-E1 transfected with the emptyexpression vector PcDNA3 and PcDNA3m ERRα, separated by SDS-PAGE (10%polyacrylamide). Panels C to M show immunolabelling for ERRα in RC cellsover a proliferation-differentiation time course: during proliferationat day 2 (C), confluence (D), nascent nodules (E), mineralized nodules(F), osteoblasts (G). Negative control for anti-rabbit antibody is shownin (H). Immunolabelling for alkaline phosphatase ALP (I), bonesialoprotein BSP (J), osteocalcin OCN (K) and osteopontin OPN (L), arealso shown. Negative control for anti-mouse antibody is shown (M).

FIG. 4, Panels A to C, show photographs of 21 days fetal calvariasections immunolabelled for ERRα. ERRα detection is seen in osteogenicfront (A), more mature growing bone trabecula (B) and remodeling bone(C). Panel D shows immunolabelling for ERRα in adult calvaria,osteocytes (D). Panels F to I show immunolabelling for alkalinephosphatase ALP (F), bone sialoprotein BSP (G), osteocalcin OCN (I) andosteopontin OPN (H). Negative controls for anti-rabbit antibody andanti-mouse antibody are shown (E,J respectively).

FIG. 5, panel A shows proliferation of RC cells treated withantisense/sense oligonucleotide at 0.5 μM, 1 μM and 2 μM (AS or S) or nooligonucleotide (Ct) during the proliferation stage between days 1-6.Inhibition of ERRα protein synthesis inhibited the cell proliferationbased on the cell number. Three 24 wells per treatment group weretrypsinized and cells were counted. Data are expressed as the cellnumber mean ±SEM and are representative of three independent experiment.ANOVA revealed a very highly significant (p<0.0001) effect of antisenseon cell proliferation. Panel B shows expression of markers of osteoblastdifferentiation. Total RNA was extracted and RT-PCR performed ontriplicate samples using specific primers for early markers (ALP, BSP,OPN, cbfa1, COLLI), proliferation (Cyclin D1, c-Fos) and apoptosis(Bcl2, Bax) at day 6. Panel C shows PCR product normalized to L32 PCRproduct. ANOVA revealed a significant (p<0.05) and a highly significant(p<0.001) effect of antisense treatment for Cbfa1 and BSP respectively.**=p<0.01, ***=p<0.001 vs control (Student's unpaired t-test).

FIG. 6, Panel A shows nodule formation in RC cells treated withantisense/sense oligonucleotide at 0.5 μM, 1 μM and 2 μM or nooligonucleotide (Ct) during the proliferation stage between days 1-6 andthen switched to normal differentiation medium. Inhibition of ERRαprotein synthesis induced a decrease in bone nodules formation Three 24wells per treatment group were von Kossa stained and the nodules werecounted. Data are expressed as the nodule mean ±SEM and arerepresentative of two independent experiment. ANOVA revealed a veryhighly significant (p<0.0008) effect of antisense, on bone nodulesformation. Panel B shows expression of osteoblast markers. Total RNA wasextracted and RT-PCR performed on triplicate samples using specificprimers for markers of osteoblast ALP, BSP, OPN, OCN, COLLI at day 15.Panel C shows PCR product normalized to L32 PCR product (C). ANOVArevealed a highly significant (p<0.001) effect of antisense treatmentfor ALP and BSP respectively. *=p<0.05, **=p<0.01, vs control (Student'sunpaired t-test).

FIG. 7, Panel A shows a Northern blot of primary RC cells transfected at50% of confluence using a pcDNA3 empty plasmid as a control andpcDNA3-ERRα at 0.5 μg of total DNA per transfection. As control, ofefficiency of the transfection, total RNA of each group was extracted 72h after transfection and northern blot was performed with samples (poolfrom three 35-mm culture). Panel B shows nodule numbers in five 35-mmdishes per treatment group for three independent experiments, von Kossastained and the nodules counted. ANOVA revealed a significant (p<0.01)effect of overexpression of ERRA, on bone nodules formation. Panel Cshows expression pattern, using specific probes for markers ofosteoblast ALP, BSP, OPN, OCN, COLLI total RNA was extracted at 72 hafter transfection, day 10 and day 13, and northern were performed (poolfrom three 35-mm culture). Data were pooled from three independentexperiments and the pattern of expression are presented.

FIG. 8, Panel A shows expression of ERRα, ERα and ERβ in primary ratcalvaria (RC) cells over a proliferation-differentiation time course byRT-PCR in presence (+Dex) or absence (−Dex) of dexamethasone (Dex)during proliferation (day 6), early nodule formation (day 10) and nodulemineralization (day 15). Total RNA was extracted and RT-PCR performedusing specific primers for ERRα, ERα and ERβ. In Panel B, PCR productwas normalized to L32 PCR product.

FIG. 9: Expression of ERRα, ERα and ERβ in primary rat bone marrowprimary culture (RBM) cells over a proliferation-differentiation timecourse by RT-PCR in presence (+Dex) or absence (−Dex) of dexamethasone(Dex) during proliferation (day 4-6), early nodule formation (day 9-11)and nodule mineralization (day 14-17) (A). Total RNA was extracted andRT-PCR performed using specific primers for ERRα, ERα and ERβ (A). PCRproduct was normalized to L32 PCR product (B).

FIG. 10 shows expression of ERRα after normalization with L32 in primaryrat bone marrow primary culture (RBM) cells over aproliferation-differentiation time course by Northern blotting inpresence of dexamethasone (Dex 10⁻⁸ M ), estrogen (E2; 10⁻⁸M), orvitamin D₃ (1.250H D3,; 10⁻⁹M) (Panel A), or PGE2 (10⁻⁹ M), TGFβ (10⁻¹⁰M) (Panel B) or 1-34 PTH (10⁻¹¹ M) (Panel C) during proliferation (day6), early nodule formation (day 10-12) and nodule mineralization (day15-16). An acute exposure of RC cells for 24 hours at beginning from day9 (nascent nodule formation) or day 15 (mature nodules present) toeither Dex, E2, D3 (A), PGE2, TGFβ (B) or 1-34 PTH (C) were also shown.

FIG. 11 shows effect of estrogen in vivo on ERRα expression level inmice. Mice were treated once weekly by subcutaneous injection witheither vehicle (0.2 ml corn oil) or 500 μg of 17β-estradiol. Total RNAfrom femur (Panel A) or flushed femur (Panel B) was extracted and RT-PCRperformed using specific primers for ERRα. ERRα PCR product wasnormalized to L32 PCR in femur and flushed femur.

FIG. 12 shows expression of ERRα in a rat model of postmenopausalosteoporosis. Female rats were either ovariectomized (OVX) orsham-operated (Sham). ERRα expression is increased 4 weeks post-surgeryin the long bones of OVX, (B, C) versus Sham-operated (A) femurs, butnot in calvaria bones (E, OVX vs F, Sham). Active osteoblasts andosteocytes (arrows, A, B) are intensely labeled as are osteoclasts(arrows, C) in sections from the OVX animals. Negative anti-rabbitantibody control is also shown (D).

DETAILED DESCRIPTION OF THE INVENTION

The present invention demonstrates a new role for the orphan receptor,estrogen related receptor alpha (ERRα), namely the modulation of boneformation in mammals.

Bone formation occurs during fetal development and post-natal growth andalso during adult life either at a low rate as part of normal boneremodelling or at an accelerated rate in response to injury or abnormalbone loss. Bone formation involves a number of processes, includingosteoblast progenitor cell proliferation, osteoblast differentiationfrom progenitor cells and mineralisation of matrix produced by theosteoblasts. The inventors have shown that ERRα plays a role in all ofthese processes.

In a rat calvaria cell culture system, which is an accepted model ofbone formation, it has been shown that upregulation of ERRα levelsincreased osteoblast differentiation and bone formation, while downregulation of ERRα led to inhibition of bone formation, with reductionof osteoblast numbers and differentiation, and aproliferation-independent, complete inhibition of both mineralised andunmineralised bone nodule formation.

ERRα has been shown in this system and in adult rat bone marrow stromalcell cultures, a second well-characterized system of osteoprogenitorcell proliferation and differentiation, to be expressed throughout allstages of osteoblastic differentiation. ERRα is more highly expressed incuboidal osteoblastic cells than in surrounding non-nodular/fibroblasticcells, and nuclear expression of ERRα increased as osteoblasts matured.Immunocytochemistry showed that ERRα is also highly expressed in vivo indeveloping fetal rat calvaria, both in sutural cells and cells at theosteogenic front, on trabecular and remodeling bone. ERRα is also highlyexpressed in fetal and adult osteocytes in calvaria and in other bonesincluding femurs.

A number of disorders are associated with bone loss or bonedegeneration. Such disorders include osteoporosis, osteoathritis,Paget's disease, periodontal disease, osteolytic bone tumour metastasesin, for example, breast cancer and multiple myeloma,osteochondrodysplasias, osteogenesis imperfecta, sclerosing bonedisplasias and osteomalacia.

The present invention provides methods and pharmaceutical compositionsfor treating such disorders to promote bone formation, by increasingERRα activity. ERRα activity may be increased in a subject either byincreasing the amount of ERRα protein present or by stimulating theactivity of existing ERRα protein. Increased ERRα activity may beachieved, for example, by up regulating expression of the ERRα gene, bygene therapy to provide a nucleotide sequence encoding ERRα protein, byadministering an agent which enhances ERRα expression, by administeringERRα protein or by administering an ERRα agonist.

Another group of diseases involves unwanted or inappropriate boneformation. Such diseases include fibrodysplasia ossificans progressive,osteoblastic bone metastases such as prostate cancer and osteosarcomas.The present invention provides methods and pharmaceutical compositionsfor treating such disorders by reducing ERRα activity. ERRα activity maybe reduced by reducing the amount of ERRα protein being produced or byinhibiting the activity of ERRα protein. This may be achieved, forexample, by administering an antisense sequence or an agent whichreduces ERRα expression, an antibody which binds specifically to ERRαprotein or an ERRα antagonist.

The invention also provides a method for screening a candidate compoundfor its ability to modulate ERRα activity in a suitable system, in thepresence or absence of the candidate compound. A change in ERRα activityin the presence of the compound relative to ERRα activity in the absenceof the compound indicates that the compound modulates ERRα activity. IfERRα activity is increased relative to the control in the presence ofthe compound, the compound is an ERRα agonist. Conversely, if ERRαactivity is decreased in the presence of the compound, the compound isan ERR antagonist.

Suitable systems for measuring ERRα activity include examination ofosteoblast proliferation or osteoblast differentiation in rat calvariacell cultures or in bone marrow stromal cell cultures as describedherein or other systems known to those of ordinary skill in the art,such as organ cultures of calvaria or femur bones or injection over thecalvaria in vivo.

In accordance with a further embodiment of the invention, the ERRαsignalling pathway may be modulated by modulating the binding of theERRα to an ERRα binding partner. Such a binding partner may include forexample the estrogen receptor. ERRα can be used to upregulate thetranscription and thus expression of genes which work together with ERRαto affect skeletal development.

The invention further provides methods for screening candidate compoundsto identify those able to modulate signaling by ERRα through a pathwayinvolving ERRα.

For example, the invention provides screening methods for compounds ableto bind to ERRα which are therefore candidates for modifying theactivity of ERRα. Various suitable screening methods are known to thosein the art, including immobilization of ERRα on a substrate and exposureof the bound ERRα to candidate compounds, followed by elution ofcompounds which have bound to the ERRα.

The invention also provides a method of modulating a ERRα signalingpathway by increasing or decreasing the availability of ERRα or bymodulating the function of the ERRα.

The invention further provides methods for preventing or treatingdiseases characterised by an abnormality in an ERRα signaling pathwaywhich involves ERRα, by modulating signaling in the pathway.

According to another aspect of the present invention is a method forsuppressing in a mammal, the proliferation of a cell capable of beingstimulated to proliferate by ERRα, the method comprising administeringto the mammal an effective amount of a ERRα antagonist or an antibodywhich binds specifically to ERRα. Such cells include but are not limitedto primitive osteoprogenitor cells.

The invention also enables transgenic non-human animal models, which maybe used for study of the effects of over and under expression of theERRα gene, for the screening of candidate compounds as potentialagonists or antagonists of this receptor and for the evaluation ofpotential therapeutic interventions.

The transgenic animals of the invention may also provide models ofdisease conditions associated with abnormalities of ERRα expression.Animal species suitable for use in the animal models of the inventioninclude mice, rats, rabbits, dogs, cats, goats, sheep, pigs andnon-human primates.

Animal models may be produced which over-express ERRα by inserting anucleic acid sequence encoding ERRα into a germ line cell or a stem cellunder control of suitable promoters, using conventional techniques suchas oocyte microinjection or transfection or microinjection into stemcells. Animal models can also be produced by homologous recombination tocreate artificially mutant sequences (knock-in targeting of the ERRαgene) or loss of function mutations (knock-out targeting of the ERRαgene). For example, knock-out targeting of the ERRα gene). For example,knock-out animal models can be made using the tet-receptor systemdescribed U.S. Pat. No. 5,654,168 or the Cre-Lox system described, forexample, in U.S. Pat. Nos. 4,959,717 and 5,801,030.

In accordance with one embodiment of the invention, transgenic animalsare generated by the introduction of a ERRα transgene into a fertilizedanimal oocyte, with subsequent growth of the embryo to birth as a liveanimal. The ERRα transgene is a transcription unit which directs theexpression of ERRα gene in eukaryotic cells. To create the transgene,ERRα gene is ligated with an eukaryotic expression module. The basiceukaryotic expression module contains a promoter element to mediatetranscription of ERRα sequences and signals required for efficient fortermination and polyadenylation of the transcript. Additional elementsof the module may include enhancers which stimulate transcription ofERRα sequences. The most frequently utilized termination andpolyadenylation signals are those derived from SV40. The choice ofpromoter and enhancer elements to be incorporated into the ERRαtransgene is determined by the cell types in which ERRα gene is to beexpressed. To achieve expression in a broad range of cells, promoter andenhancer elements derived from viruses may be utilized, such as theherpes simplex virus thymidine kinase promoter and polyoma enhancer. Toachieve exclusive expression in a particular cell type, specificpromoter and enhancer elements could be used, such as the promoter ofthe mb-1 gene and the intronic enhancer of the immunoglobulin heavychain gene. In a preferred embodiment, a bone specific promoter such asthe bone sialoprotein promoter may be used to target expression inosteoblasts.

The ERRα transgene is inserted into a plasmid vector, such as pBR322 foramplification. The entire ERRα transgene is then released from theplasmid by enzyme digestion, purified and injected into an oocyte. Theoocyte is subsequently implanted into a pseudopregnant female animal.Southern blot analysis or other approaches are used to determined thegenotype of the founder animals and animals generated in the subsequentbackcross and intercross.

Such deficient mice will provide a model for study of the role of ERRαin bone cell differentiation and proliferation and general skeletaldevelopment. Such animals will also provide tools for screeningcandidate compounds for their interaction with ERRα or the signallingpathway activated by ERRα.

The invention also provides pharmaceutical compositions for promotingbone formation, comprising as active ingredient a substantially purifiedERRα protein, an ERRα agonist or an isolated nucleotide sequenceencoding ERRα protein.

Such compositions are useful, for example, in treating disordersassociated with bone loss.

ERRα protein may be produced by conventional recombinant techniquespermitting expression of ERRα by a suitable host cell. A DNA encodingERRα may be prepared as described, for example, in Giguere et al.(1998). Techniques for production of proteins by recombinant expressionare well known to those in the art and are described, for example, inSambrook et al. (1989) or latest edition thereof. Suitable host cellsinclude E. coli or other bacterial cells, yeast, fungi, insect cells ormammalian cells.

The invention provides for compositions for promoting bone formationcomprising as active ingredient an ERRα agonist obtained by using ascreening method as described herein.

It may be advantageous, in treating disorders associated with bone loss,to employ a combination therapy, first administering an agent such as abiphosphonate to suppress osteoclast function, followed after a suitableperiod of time, by administration of a pharmaceutical composition forpromoting bone formation, as described herein. Such treatment regimensare well known to those of ordinary skill in the art.

A nucleotide sequence encoding ERRα protein may be administered to asubject experiencing bone loss due to an absent or defective ERRα geneeither in vivo or ex vivo. Expression may be targeted to a selected cellor tissue by use of an appropriate promoter, for example thebone-specific promoter for bone sialoprotein (Stein et al. (2000)). Forexample, stem cells or bone marrow stromal cells may be obtained from asubject and treated in vivo with the nucleotide sequence, the cells thenbeing restored to the subject. Such methods are described in Horwitz etal. (1999).

The invention also provides pharmaceutical compositions for reducingbone formation, comprising as active ingredient an antibody which bindsspecifically to ERRα, an ERRα antagonist or a negative regulator such asan antisense nucleic acid or a dominant negative mutant version of theERRα gene.

The invention provides for compositions for reducing bone formationcomprising as active ingredient an ERRα antagonist obtained by using ascreening method as described herein.

Antibodies which bind specifically to ERRα protein may be made byconventional techniques.

The term “antibodies” includes polyclonal antibodies, monoclonalantibodies, single chain antibodies and fragments such as Fab fragments.

In order to prepare polyclonal antibodies, fusion proteins containingdefined portions or all of an ERRα protein can be synthesized inbacteria by expression of the corresponding DNA sequences, as describedabove. Fusion proteins are commonly used as a source of antigen forproducing antibodies. Alternatively, the protein may be isolated andpurified from the recombinant expression culture and used as source ofantigen. Either the entire protein or fragments thereof can be used as asource of antigen to produce antibodies.

The purified protein is mixed with Freund's adjuvant and injected intorabbits or other appropriate laboratory animals. Following boosterinjections at weekly intervals, the animals are then bled and the serumisolated. The serum may be used directly or purified by various methodsincluding affinity chromatography to give polyclonal antibodies.

Monoclonal anti-ERRα antibodies may be produced by methods well known inthe art. Briefly, the purified protein or fragment thereof is injectedin Freund's adjuvant into mice over a suitable period of time, spleencells are harvested and these are fused with a permanently growingmyeloma partner and the resultant hybridomas are screened to identifycells producing the desired antibody. Suitable methods for antibodypreparation may be found in standard texts such as Barreback, E. D.(1995).

The pharmaceutical compositions of the invention may comprise, inaddition to the active ingredient, one or more pharmaceuticallyacceptable carriers.

Administration of an effective amount of a pharmaceutical composition ofthe present invention means an amount effective, at dosages and forperiods of time necessary to achieve the desired result. This may alsovary according to factors such as the disease state, age, sex, andweight of the subject, and the ability of the composition to elicit adesired response in the subject. Dosage regima may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

By pharmaceutically acceptable carrier as used herein is meant one ormore compatible solid or liquid delivery systems. Some examples ofpharmaceutically acceptable carriers are sugars, starches, cellulose andits derivatives, powdered tragacanth, malt, gelatin, collagen, talc,stearic acids, magnesium stearate, calcium sulfate, vegetable oils,polyols, agar, alginic acids, pyrogen-free water, isotonic saline,phosphate buffer, and other suitable non-toxic substances used inpharmaceutical formulations. Other excipients such as wetting agents andlubricants, tableting agents, stabilizers, anti-oxidants andpreservatives are also contemplated.

The compositions described herein can be prepared by known methods forthe preparation of pharmaceutically acceptable compositions which can beadministered to subjects, such that an effective quantity of the activesubstance is combined in a mixture with a pharmaceutically acceptablecarrier. Suitable carriers and formulations adapted for particular modesof administration are described, for example, in Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., USA 1985). On this basis thecompositions include, albeit not exclusively, solutions of the substancein association with one or more pharmaceutically acceptable vehicles ordiluents, and contained in buffered solutions with a suitable pH andiso-osmotic with the physiological fluids.

The pharmaceutical compositions of the invention may be administeredtherapeutically by various routes such as by injection or by oral,nasal, buccal,. rectal, vaginal, transdermal or ocular routes in avariety of formulations, as is known to those skilled in the art.

The present invention also enables the analysis of factors affecting theexpression of the ERRα gene in humans or in animal models. The inventionfurther provides a system for screening candidate compounds for theirability to turn on or turn off expression of the ERRα gene and theidentification of binding partners which may also affect expression ofERRα or certain downstream partners.

For example, an RC cell culture system can be used to identify compoundswhich activate production of ERRα or, once ERRα production has beenactivated in the cells, they can be used to identify compounds whichlead to suppression or switching off of ERRα production. Alternatively,such a cell culture system can be used to identify compounds or bindingpartners of ERRα which increase its expression. Compounds thusidentified are useful as therapeutics in conditions where ERRαproduction is deficient or excessive.

The present invention enables also a screening method for compounds oftherapeutic utility as antagonists of the biological activity of ERRα.Such antagonist compounds are useful, for example, to reduce or preventdifferentiation and maturation of osteoblasts and osteocytes. ERRαantagonists may also be used in the treatment of bone related disordersinvolved inappropriate bone cell growth. Those skilled in the art willbe able to devise a number of possible screening methods for screeningcandidate compounds for ERRα antagonism.

A screening method may also be based on binding to the ERRα receptor.Such competitive binding assays are well known to those skilled in theart. Once binding has been established for a particular compound, abiological activity assay is employed to determine agonist or antagonistpotential.

ERRα is Expressed in Osteoblast Lineage Cells Throughout TheirDevelopmental Lifetime and ERRα mRNA and Protein are More Highly andWidely Expressed than Either ERα and ERβ.

The inventors have shown that ERRα mRNA is expressed in differentiatingprimary cultures of RC cells and rat bone marrow stromal (RBM) cells andin single isolated osteoblast colonies at all detectable stages ofdifferentiation, suggesting that ERRα may have a function in osteoblaststhroughout their developmental lifetime. ERRα mRNA is present at muchhigher levels than either ERα or ERβ mRNA in both RC and RBM cellcultures. ERRα protein was also found more widely distributed in vitroin RC cell cultures than either ERα or ERβ. ERRα was found in most ifnot all cells in RC cell cultures from early proliferation stagesthrough mineralized nodule formation. ERα was also detected in RC cellsat all times analysed but at lower levels than ERRα which is also thecase in rat bone marrow cultures (data not shown). ERβ, on the otherhand, was more difficult to detect at any time other than in earlyproliferating cultures. These observations fit with the expressionpattern of these three receptors in vivo in 21 day fetal calvaria.Indeed, ERRα is more highly and widely expressed than either ERα or ERβ,being highly expressed in sutural cells and all identifiable osteoblastsand osteocytes. ERα is not highly expressed in nascent, but isdetectable in more mature, osteoblastic cells. ERβ, on the other hand,is more highly expressed in sutural and nascent osteoblasts. Theseresults suggest that ERRα and ERα and/or ERβ are co-expressed in atleast some osteoblastic cells, and that these receptors may act alone ortogether to regulate the expression of target genes in bone. It is alsonotable that ERRα protein is localized primarily in either the nucleusor the cytoplasm or both depending on the developmental stage of theosteoblast. This suggests that ERRα target genes and function may varydepending on the maturational stage of the osteoblastic cells.

In addition to its expression in fetal calvaria, ERRα is also highlyexpressed in adult calvaria and other fetal and adult bones, includinglong bones such as the femur. It is also throughout osteogenesis inadult rat bone marrow stromal cell cultures, suggesting that it mayfunction throughout the lifetime of the organism and in all bones of thebody irrespective of the developmental process by which they form, i.e.,through an intramembranous or endochondral route. In adult quiescentbone, labeling appears highest in osteocytes, which are thought to bemechanosensors that send strain-related signals to lining cells locatedat the bone surface through the canicular syncytium (Huiskes et al,2000), leading to recruitment of osteoblasts amongst other effects.These data suggest that ERRα may function not only during fetal bonedevelopment but also in adult life in both bone formation. andmaintenance.

Previously, it has been shown that the estrogen receptors ERα and ERβare themselves expressed in osteoblasts and osteocytes (Braidman et al,1995; Onoe et al, 1997; Windahl et al, 2000), raising the possibilitythat ERRα and one or both of the ERs may be co-expressed in at leastsome osteoblastic cells. The inventors have shown by both mRNA andprotein analysis that ERRα and ERα are co-distributed in large cohortsof osteoblastic cells, raising the possibility that these receptors mayregulate the expression of the same target genes in bone via their knownability to participate in protein-protein interactions (Johnston et al,1997) and their recently described capacity to bind to the same DNAtarget (SFRE and ERE) sequence on the osteopontin promoter (Vanacker etal, 1999).

Together, these data suggest that ERRα, ERα and perhaps ERβ areco-expressed in osteoblastic cells, and may display at least somefunctions in common, either singly or through their interactions, withregulatory capacities to act on target genes.

ERRα and Proliferation

Consistent with its expression in proliferating osteoblasticpopulations, we have found that antisense oligonucleotide-induceddownregulation of ERRα inhibits proliferation of RC cell populations, aninhibition that appears to have consequences on bone nodule formation atlater times (see below). The decrease in proliferation was somewhatunexpected, given our previous observation that ERRα expression appearsto correlate with exit from proliferation and the onset of thedifferentiation process in at least certain other cell types, includingthe nervous system, the epidermis and muscles in the developing mouse(Bonnelye et al, 1997). This suggests that ERRα may play cell-typespecific functions and is in keeping with its detection from the onsetof osteogenesis in vivo (Bonnelye et al, 1997) and its presence in allosteoblastic cells including the earliest detectable osteoprogenitors(current data).

The molecular basis for the ERRα effect on proliferation is of interest.Since OPN has been described as a target gene of ERRα in in vitropromoter-reporter assays (Bonnelye et al, 1997; Vanacker et al, 1998),and since OPN is highly expressed in many proliferative populationsincluding osteoprogenitors (FIG. 2) and in many tumour cell lines (seereview in Denhardt and Noda, 1998), one candidate target in theproliferation time window for antisense-induced downregulation was OPN.However, we found no detectable downregulation of this molecule duringthis developmental time window, although it is clearly sensitive toregulation by ERRα later during the differentiation phase of thecultures (see below). We also found no significant changes in theantisense-treated RC populations in expression of a variety ofproliferation and apoptosis/survival-associated genes expressed inosteoblasts including c-fos, Bcl-2, and Bax. However, we did observe asignificant decrease in cyclin D1, a regulator of G1 phase progression.Interestingly, estrogen induces cell proliferation by stimulatingprogression through the G1 phase of the cell cycle (Clarke et al, 1992;Wakeling et al, 1991), and induction of cyclin Dl expression is acritical feature of the mitogenic response to estrogen. There is also astrong correlation between increased levels of cyclin D1 mRNA withestrogen receptor overexpression in breast cancer cells (Buckley et al,1993). Recently, Sabbah et al., have described a region in the cyclin D1promoter that confers regulation by estrogens in the human mammarycarcinoma cells MCF7. The induction is strictly hormone dependant andrequires the DNA binding domain as well as both AF-1 and AF-2 domains ofERα (Sabbah et al, 1999). Although no ERE has been identified in thecyclin D1 promoter, it is possible that ERα activates cyclin D1transcription by its ability to heterodimerize with c-jun/ATF-2 (Sabbahet al, 1999). ERRα has also been described as a modulator of theestrogen receptor-mediated response of the human lactoferrin genepromoter (Yang et al., 1996), a mechanism that may also underlie itsability to regulate cyclin D1.

ERRα, Osteoblast Differentiation and Matrix Mineralization

The findings described herein show a. critical role for ERRα in boneformation, with both up- and down-regulation of bone nodule formationconcomitant with up- and down-regulation of ERRα expression in vitro.Up-regulation of ERRα by transfection of RC cells with a full-lengthERRα expression vector late in the proliferation time window increasedbone nodule formation by an amount approximately equivalent to thetransfection efficiency of the population. Concomitantly, all bonemarkers expressed at early differentiation stages (ALP, OPN, BSP andCOLLI; Aubin and Liu, 1996) were upregulated 72 h after transfection;OCN, a late marker of the mature osteoblast, was also upregulated atdays 10 and 13. Whether the increase in osteoprogenitor differentiationand bone nodule formation is a consequence of upregulation of any ofthese bone markers, or results from regulation of another currentlyunknown ERRα target gene, remains to be explicitly tested.

Downregulation of ERRα also had marked inhibitory effects ondifferentiation and bone nodule formation, when it was downregulatedduring proliferation phase or earlier or later in the differentiationsequence. When RC cells were treated with antisense oligonucleotidesonly during the proliferation window (day 1-6) and then returned tonormal medium, the number of bone nodules present at day 15 was reducedcompared to untreated or sense-treated cells. While one can speculatethat this decrease reflects the downregulation of cyclin D1 anddecreased proliferation of osteoprogenitors amongst other cells, thedecrease could also reflect the concomitant downregulation of the bone“master” gene Cbfa1 (Ducy et al, 1997; Komori et al, 1997) and BSP, bothof which are upregulated early during osteoprogenitor celldifferentiation (Aubin and Liu, 1996; Malaval et al., 1999); we alsofound that BSP and ERRα are co-expressed in these very earlyosteoprogenitors (FIG. 2). The latter possibility is consistent with thefinding that downregulation of ERRα only after proliferation has largelyceased (antisense treatment from day 5-11) results in completeinhibition of mineralized bone nodule formation, and concomitantdownregulation of cbfa1, BSP and OCN. These observations, together withthe data on increased bone formation when ERRα is upregulated early,suggest that at least part of the effect of ERRα on osteoblastdifferentiation and bone formation occurs early during thedifferentiation sequence, such that differentiation may not progressbeyond a certain point when ERRα levels are low. In keeping with thishypothesis, large flat but ALP-positive colonies are present inantisense-treated cultures and a few cells express diminished levels ofother osteoblast markers (FIG. 8).

It is also notable that ERRα also plays a role late in thedifferentiation/maturation sequence, i.e., when matrix is mineralizing.When RC cells were treated with antisense during latedifferentiation-matrix mineralization stages (day 9-15), we observed aless pronounced but nevertheless dose-dependent decrease in mineralizednodule number, but those that did form appeared to cover a largersurface area (i.e., more bone was deposited per colony) than those incontrol cultures.

ERRα Expression is Stimulated By Estrogen In Vitro and In Vivo and isUpregulated in the OVX Rat Model of Postmenopausal Osteoporosis

Estrogen (17β-estradiol; E2) was found to regulate ERRα at early timesin chronically treated RC cell cultures, while an acute (24 h) treatmentat either day 9 or day 15 did not. These results suggest a link betweenERRα and E2 in bone, most likely during the proliferation phase. Shigetaet al. showed that E2 can also activate ERRα in the uterus (Shigeta etal, 1997). Importantly, we found that 17β-estradiol also upregulatesERRα in bone in vivo. Based on the kinetics of upregulation after E2administration, this regulation appears to be an immediate or acuteresponse to the administration of E2. Together, these results suggest alink between ERRα and estrogens in two estrogen-sensitive tissues.

ERRα and ERα are both expressed in adult osteocytes in calvaria and longbones suggesting a function of ERRα during adult life. Bone loss in theaging skeleton is accelerated by a decrease in secretion of estrogens inpost-menopausal women and can be reversed by administration of naturalor synthetic estrogens. The decrease in estrogen also induces a decreasein the expression of ERα (Hoyland et al, 1999). Given these data, it wassurprising to measure an increase in ERRα in OVX rats, although theacute nature of the response in vitro to estrogen supplementation,similar to the acute upergulation in vivo which was followed bydownregulation, may help to explain the discrepancy. However, it is alsoworth noting that ERRα is highly expressed in the osteoblasts present inthe high turnover bone of the OVX rats, which may indicate that ERRαexpression is essential for osteoblast function in osteoporosis. Thedata are consistent with E2 having a biphasic effect on ERRα expressionin bone.

ERRα Expression is Stimulated by TGFβ and Vitamin D3, Decreased ByPTH-1-34 and Unaffected By Dexamethasone in Proliferating RC CellCultures

ERRα is also regulated during the proliferation stages of RC cellcultures by another hormone (1,25(OH)₂D₃) and a growth factor (TGFβ withpotent regulatory activities in bone metabolism. Acute treatment with1,25(OH)₂D₃ or TGFβ for 24 hours later in the developmental sequence (atday 9 or day 15) had no effect on ERRα expression. It will therefore beof interest to determine whether 1,25(OH)₂D₃ and TGFβ effects may bemediated at least in part by its abilities to modify ERRα levels. Therewas also a down regulation of ERRα by PTH-1-34 during proliferationwhich was most evident late in RC cultures when mineralized nodules arepresent. However, acute treatment with PTH-1 -34 for 24 hours at day 7or day 16 did inhibit the expression of ERRα which suggests a moredirect effect of PTH-1-34 on ERRα expression compared to E2, 1,25(OH)₂D₃or TGFβ.

ERRα expression in RC and RBM cell populations is almost the same incells grown without (−Dex) or with (+Dex) dexamethasone. A 24 hour acutetreatment of RC cells with Dex at day 9 or day 15 also had no effect onthe ERRα expression level. These results suggest that, in RC and RBMcell populations, Dex has no effect on the expression of ERRα while itdoes downregulate ERα and even more potently downregulates ERα, suggestdifferential regulation by glucocorticoids of ERRα versus the ERs.

EXAMPLES

The examples are described for the purposes of illustration and are notintended to limit the scope of the invention.

Methods of chemistry, molecular biology, protein and peptidebiochemistry and immunology referred to but not explicitly described inthis disclosure and examples are reported in the scientific literatureand are well known to those skilled in the art.

The following materials and methods were employed in the Examples whichfollow.

Cell Culture

Cells were enzymatically isolated from the calvaria of 21 d Wistar ratfetuses by sequential digestion with collagenase as described previously(Bellows, 1986). Cells obtained from the last four of the five digestionsteps (populations II-V) were pooled and plated in T-75 flasks in α-MEMcontaining 15% heat-inactivated FBS (Flow Laboratories, McLean, Va.) andantibiotics comprising 100 mg/ml penicillin G (Sigma Chemical Co., St.Louis, Mo.), 50 mg/ml gentamycin (Sigma), and 0.3 mg/ml fungizone (FlowLaboratories). After 24 h incubation, attached cells were washed withPBS to remove nonviable cells and other debris, and then collected bytrypsinization using 0.01% trypsin in citrate saline. Aliquots werecounted with a Coulter Counter (Coulter Electronics, Hialeah Fla.), andthe remaining cells were resuspended in the standard medium describedabove. The resuspended cells were plated into 100 mm tissue culturedishes at 10⁵ cells/dish, into 35 mm tissue culture dishes at 2×10⁴/dishand in 24 wells plates at 10⁴ cells/well. After 24 h incubation, mediumwas changed and supplemented with 50 μg/ml ascorbic acid, 10 mM sodiumβ-glycerophosphate, and with or without 10⁻⁸ M dexamethasone (Merck,Sharp, and Dohme, Canada, Ltd., Kirkland, PQ), or 10⁻⁸ M 17β-estradiol(E2; Sigma), or 10⁻⁹ M 1,25(OH)₂D₃, or 10 M PGE₂, 10⁻¹⁰ M TGFβ or 10⁻¹¹M 1-34 PTH (Sigma). Medium was changed every 2 days. All dishes wereincubated at 37° C. in a humidified atmosphere in a 95% air/5% CO₂incubator.

Bone marrow stromal cells from the femurs of young male Wistar rats,110-120 g body weight, were cultured essentially as described (Aubin etal, 1998). The rats were killed by cervical dislocation, the femurs weredissected under aseptic conditions and placed in medium (MEM) containingantibiotics (1 mg/ml penicillin G (Sigma Chemical Co., St. Louis, Mo.),500 μg/ml gentamycin sulfate (Sigma) and 3 μg/ml fungizone (FlowLaboratories, McLean, Va.) (designated 10X AB)). The adherent connectivetissue and muscles were removed, the femurs were placed in fresh 10X AB,and their ends (epiphyses and metaphyses) were cut off with a scalpel.With a 22 gauge needle and syringe, 5 ml of MEM were flushed througheach femur until the bone appeared blanched (about five to eight times).The resulting cell suspension was flushed through a syringe severaltimes to produce a largely single cell suspension; cells recovered fromtwo femurs were added to a T-75 tissue culture flask (Falcon) andincubated in a 37° C. humidified 95% air/5% CO₂ incubator. Growthmedium, consisting of MEM containing 10% fetal calf serum, antibiotics(100 μg/ml penicillin G, 50 μg/ml gentamycin and 0.3 μg/ml fungizone),50 μg/ml ascorbic acid, and 10⁻⁸ M dexamethasone (Sigma), was changedevery 2-3 days. After 7 days, cells in each T-75 cell culture flask werewashed with 15 ml of warm PBS and adherent cells were recovered with amixture of 3 ml of 0.2% trypsin (w/v in citrate saline) and 2 ml ofcollagenase. Recovered cells were passed through a syringe with a 22gauge needle to insure a single cell suspension. Cells were then countedon a Coulter Counter (Coulter Electronics, Hialeah Fla.) and plated atdensities between 5×10³ and 2×10⁴ cells/35 mm dish and 2×10⁵ cells/100mm dish (Falcon). Cells were cultured in αMEM supplemented as above andchanged every 2-3 days, for approx. 17 days until mature bone noduleswere seen. To promote mineralization, 10 mmol/ml of β-glycerophosphate(Sigma) was added for at least 2 days of culture prior to fixation.Cultures were then stained and the colonies quantified as indicated inwhat follows.

Northern Blots

Total RNA was extracted with guanidine from RC cells at different timesof the culture corresponding to different stages of proliferation,differentiation and bone nodule formation (Current Protocols inMolecular Biology, vol. 1, 1996). Northern blots were prepared andhybridized with a 750bp fragment corresponding to the rat 3′ UTR of ERRαaccording to standard procedures (Chirgwin et al, 1979). Rat α1 COLL-I(Genovese et al.) was a 900bp cDNA PstI fragment containing the entire3′ noncoding region and one-half of the C-terminal of the propeptide ofthe α1 chain of type I. Rat bone/liver/kidney ALP (Noda et al., 1987)was a 600 bp cDNA EcoRI fragment obtained by digesting pRAP54 withBssHII-XhoI to remove 1.8 kb of the 5′ region and religating the bluntends. Rat OPN was a 700 bp cDNA BamHI-EcoRI fragment obtained bydigesting the full length cDNA with PvuI to remove 800 bp of 5′ regionand ligating the blunt ended fragment into SmaI cut pGEM-7Zf(+) vector(Promega, Madison, Wis.). Rat OCN was a partial cDNA containing 350 bpof the 3′ UTR isolated with OCN-specific primers from a λgt11 libraryprepared from ROS 17/2.8 cells, rat BSP was a partial cDNA containing500bp of 3′ region isolated with BSP-specific primers from a λgt11library prepared from RC cells forming bone nodules, and rat L32 wasgenerated from RC cell mRNA by PCR using specifics primers; theidentities of the OCN, BSP and L32 probes were confirmed by sequencing(Liu et al., 1994).

RT-PCR

Samples of total cellular RNA (1.5-5 μg) were reverse-transcribed usingoligo dT and the first strand synthesis kit of Superscript™II, GibcoBRL. PCR was performed with specific primers specific for ERRα. Primers,located in different exons, were as follows: ERRα upstream CAG GAA AGTGAA TGC CCA GG (3′UTR): ERRα downstream CTT TGC AGC AAA TAT ACA TT(3′UTR): ERα upstream GAG CTG CCA ACC TTT GGC CAA GT (Dom D 5′): ERαdownstream TGA ACT TGA TCG TGG AGA TTC (Dom D 3′): ERβ upstream AAA GCCAAG AGA AAC GGT GGG CAT (Dom D): ERβ downsteam GCC AAT CAT GTG CAC CAGTTC CTT (Dom E): L32 upstream: CAT GGC TGC CCT TCG GCC TC L32downstream: CAT TCT CTT CGC TGC GTA GCCThe PCR reaction mixture contained cDNA (1 μl), 1 μl dNTP mix (20 mM),10× PCR buffer, Q solution, 25 pmol primers and 5 Units of Taqpolymerase from Quiagen. PCR was done for 25 cycles (94° C. for 1 min,55° C. for 1 min, 72° C. for 1 min and, a final elongation step of 7 minat 72° C.) for ERRα and L32; 35 cycles (94° C. for 1 min, 55° C. for 1min, 72° C. for 1 min and a final elongation step of 7 min at 72° C.)for ERα; 45 cycles (94° C. for 1 min, 59° C. for 1 min, 72° C. for 1 minand a final elongation step of 7 min at 72° C.) for ERβ. Amplimers weresequenced for verification.

Osteoblast-associated and other markers were also amplified by PCR usingspecific primers for rat OCN, OPN, ALP, BSP, Cbfa1, COLL I (collagentype I α chain), C-fos, Cyclin D1, Bax and Bcl-2. PCR was done for 25cycles (94° C. for 1 min, 55° C. for 1 min, 72° C. for 1 min and a finalelongation step of 7 min at 72° C.) for OCN, OPN, ALP, BSP, L32, Bax and30 cycles for Bcl-2, 32 cycles for c-Fos, 35 cycles for Cyclin D1 andCbfa1 (with annealing temperatures of 58° C. and 62° C. respectively and23 cycles for COLLI with annealing temperature of 59° C. OC upstream:AGG ACC CTC TCT CTG CTC AC OC downstream: AAC GGT GGT GCC ATA GAT GC BSPupstream: CGC CTA CTT TTA TCC TCC TCT G BSP downstream: CTG ACC CTC GTAGCC TTC ATA G ALP upstream: CCC GCA TCC TTA AGG GCC AG ALP downstream:TAG GCG ATG TCC TTG CAG C OPN upstream: GCC ACT TGG CTG AAG CCT G OPNdownstream: GAA ACT CCT GGA CTT TGA CC CbfA1 upstream: CTT CAT TCG CCTCAC AAA C CbfA1 downstream: CAC GTC GCT CAT CTT GCC GG Cyclin D1upstream: TCC CGC CAG CAG CAA GAC AC Cyclin D1 downstream: TGA GCT TGTTCA CCA GAA GC c-Fos upstream: ATA GAG CCG GCG GAG CCG CG c-Fosdownstream: AAG CCC CGG TCG ACG GGG TG Bax upstream: CCT TGG AGC AGC CGCCCC AG Bax downstream: ATG TGG GCG TCC CGA AGT AGG Bcl-2 upstream: GGGGAA ACA CCA GAA TCA AG Bcl-2 downstream: AGA GAA GTC ATC CCC AGC CCCOLLI upstream: GGA GAG AGT GCC AAC TCC AG COLLI downstream: CCA CCC CAGGGA TAA AAA CTPoly (A) PCR Library Selection

Nineteen poly(A)PCR libraries representative of five transitional stagesin osteoblast lineage progression were selected from more than onehundred available amplified colonies (Liu et al., 1994; Liu, F andAubin, J. E., submitted). Stage A are replica-plated monolayer coloniescommitted to differentiate to the osteoblast lineage but not yetexpressing type I α1 collagen or alkaline phosphatase, both earlymarkers of osteoprogenitor cells. Stage B and C colonies areprogressively more mature, i.e. expressing type I α1 collagen or bothtype I α1 collagen and alkaline phosphatase respectively. Stage Dcolonies represent multilayered cells and contain histologicallyidentifiable cuboidal osteoblasts. Stage E colonies comprised terminaldifferentiation stages, with multilayered cells and mineralized bonematrix. Relative amounts of total cDNA were determined by Southernhybridization and were used for normalization.

Western Blots

Total protein was extracted from confluent HeLa and MC3T3-E1 cellsaccording to standard methods (Current Protocols in Molecular Biology,vol. 1, 1996). Western blot analyses were performed using a semi-drysystem. Immunoblotting was performed with rabbit polyclonal antiserumprepared against a rat peptide (NH-CPASDECEITKRR-C) localized in the Cdomain of ERRα; blots were incubated overnight at room temperature withthe antiserum diluted to 1/500, and binding was detected usingHRPO-conjugated goat-anti-rabbit antibodies (1/3000; BioRad) andchemoluminescence.

Immunolabelling

Immunolabelling of cultures was done essentially as described previously(Turksen, 1991; Turksen, 1992). Cultures were rinsed with PBS, fixedwith 3.7% formaldehyde in PBS and permeabilized with methanol at −20° C.Frozen sections were fixed 10 min in cold acetone. Paraffine sectionswere treated deparaffined in xylene, then rehydrated in 100%, 95% and70% ethanol and water. After rinsing, cells in dishes or frozen,paraffine sections were incubated for 1 h at room temperature with 10%normal serum in PBS for ERRα and ERα and in 3% BSA in PBS (denaturated)for ERβ OCN, ALP, OPN, and BSP. After rinsing, cells or sections wereincubated for 1.5 hours with appropriate dilutions of primary antibodies(1/50, anti-ERRα; anti-ERα or anti-ERβ (MC-20 or Y-19, respectively;Santa Cruz Biotechnology, Inc) )(Shim et al, 1999; Saji et al, 2000;Tremblay et al 1999). The anti-rat OCN antiserum was kindly provided byDominique Modrowski (INSERM U349, Hopital Lariboisiere, Paris, France)and used at 1/100 dilution. The anti-OPN MPIIIB10) and anti-BSP (WWVIDI)antibodies were purchased from the Hybridoma Bank (Iowa City, Iowa) andused at a 1/800 and 1/500 dilution respectively. The production andcharacterization of monoclonal antibody RBM 211.13 directed to ratbone/liver/kidney ALP, have been described elsewhere (Turksen and Aubin,1991; Turksen et al., 1992); it was used at a 1/100 dilution of purifiedascites fluid. 10% normal serum in PBS or 3% BSA in PBS were used asnegative controls. Nodules or calvaria sections were rinsed in PBS andincubated for 1 h at room temperature with secondary antibodyCY-3-conjugated anti-rabbit (Jackson lmmunoresearch Lab, West Grove,Pa., USA; 1/300 final dilution) for ERRα, OC and ALP or secondaryantibody anti-mouse (Jackson lmmunoresearch Lab, West-Grove, Pa., USA;1/300 final dilution) for BSP and OPN. After rinsing, samples weremounted in Moviol (Hoechst Ltd, Montreal, PQ, Canada) and observed byepifluorescence microscopy on a Zeiss Photomicroscope III (Zeiss,Oberkochen, Germany). For photography and printing, equal exposure timeswere used for specifically-labelled and control cultures.

Nodule Quantification

For quantification of nodule formation, dishes or wells were fixed andstained by the Von Kossa technique and bone nodules were counted on agrid (Bellows et al., 1986; Bellows and Aubin, 1989). Results areplotted as the mean number of nodules±SD of three wells for controls andeach concentration of antisense or sense primers and five dishes forpcDNA3 control and pcDNA3-ERRα.

Cell Counting

For cell growth analysis, the cell layers were rinsed in PBS, releasedwith trypsin and collagenase (1:1, vol/vol, of solutions describedabove), and the harvested cells were counted electronically. Results areplotted as the average of three counts for each of three dishes forcontrol and pcDNA3-ERRα or three wells for each concentration ofantisense or sense primers used.

Alkaline Phosphatase Histochemistry

The histochemical stain for alkaline phosphatase is a modification ofPearse's (1960). Cells were rinsed once with cold PBS and fixed in 10%cold neutral buffered formalin for 15 min, rinsed with distilled water,and left in distilled water for 15 min. Fresh substrate (10 mg NaphtholAS MX-PO4 (Sigma) dissolved in 400 μl N,N-dimethylformamide, then addedto 50 ml distilled water and 50 ml Tris-HCl (0.2 M, pH 8.3) and then 60mg Red Violet LB salt (Sigma)), was filtered through Whatman's No. 1filter directly onto the dishes, and incubated for 45 min at 20° C. Thedishes were then rinsed in tap water, drained and stained with 2.5%silver nitrate for 30 min at room temperature (von Kossa stain), thenrinsed 3 times with tap water. The dishes were finally air dried.

Transient Transfections

Primary RC cells were grown in 35 mm tissue culture dishes at 2 10⁴/dishin α-MEM containing 10% heat-inactivated FBS (Flow Laboratories, McLean,Va.) and supplemented with 50 μg/ml ascorbic acid, 10 mM sodiumβ-glycerophosphate, and 10⁻⁸ M dexamethasone. Cells were transfected at50% of confluence according to the Effecten transfection protocol(Quiagen) using a pcDNA3 empty plasmid as a control and pcDNA3-ERRα (inthe EcoRI cloning site) at 0.5 μg of total DNA per transfection. Ascontrol of transfection efficiency, we used a CMV-βGal vector. Noduleswere counted at day 15. mRNA was extracted at 72 h, day 10 (beginning ofnodules formation) and day 15 (mineralized nodules), after transfection.

Antisense and Sense Oligonucleotide Treatment

The resuspended RC cells were plated in 24 wells plates at 10⁴cells/well. Antisense oligonucleotide inhibition of ERRα expression wasaccomplished with a 20-base phosphorothioate-modified oligonucleotide,localized to the A/B domain. The ERRα antisense oligonucleotide sequencewas: 5′-TCACCGGGGGTTCAGTCTCA-3′. Control dishes were treated with thecomplementary sense oligonucleotide or no oligonucleotide. Preliminaryexperiments were done to determine effective oligonucleotideconcentrations that were not toxic. 0.1 μM to 5 μM oligonucleotides wereadded directly to cells either during the proliferation phase (days 1 to6) and 0.5 μM to 2 μM oligonucleotides were added during thedifferentiation phase (day 5 (end of proliferation) to 11) or (day 9(nascent nodule formation) to 15) in standard medium as abovesupplemented with 50 μg/ml ascorbic acid, 10 mM sodiumβ-glycerophosphate, and 10⁻⁸ M dexamethasone. Medium was changed every 2days and fresh oligonucleotides were added. mRNA was collected at day 6for the proliferation experiments and at day 15 for the differentiationexperiments. Nodules were counted at 15 days.

Ovariectomized Rats and Estrogen-treated Mice

8 week-old female CBA-1 mice from the University of Bristol breedingcolony were treated once weekly by subcutaneous injection with eithervehicle (0.2 ml corn oil; Sigma Chemical, Poole, Dorset, U.K.) or 500 μgof 17β-estradiol (Sigma) as described (Samuels et al, 1999). This dosewas chosen because it has previously been reported to cause maximalstimulation of endosteal bone formation in female mice (Brain et al,1993). Throughout the experiment, animals received a standard diet (ratand mouse standard diet, B&K Ltd., Humbside, U.K.) and water ad libitum,and were kept with a cycle of 12 h light and 12 h darkness. Animals weresacrificed 4, 8, 12 and 24 days after the first subcutaneous injection,the tibia were removed for processing, and mRNA was extracted (Samueland Tobias, 1999).

35-40 day old (100-125 g) Wistar rats (Charles River BreedingLaboratories, Quebec), either ovariectomized (OVX) or sham-operated(SHAM), were kept under standard diet and laboratory conditions for 4weeks post-operation. Animals were killed, the uteri were dissected andweighed, and the femurs were removed, fixed in 4% paraformaldehyde,decalcified for 2 weeks in EDTA and processed overnight for paraffinembedding.

Example 1

ERRα mRNA and Protein are Expressed Throughout All OsteoblastProliferation and Differentiation Stages In Vitro

ERRα mRNA expression levels were assessed over aproliferation-differentiation time course by Northern blotting ofprimary rat calvaria (RC) cell populations grown in the presence (+Dex)or absence (−Dex) of dexamethasone (Dex), a stimulator ofdifferentiation in this model. Under both growth conditions, ERRα mRNAwas expressed at all times assessed, including proliferation (day 6),early nodule formation (day 10) and nodule mineralization (day 15) (FIG.1A, B). For comparison, mRNA levels for three osteoblast markers,alkaline phosphatase (ALP; a relatively early marker of osteoblastdevelopment), osteopontin (OPN) and osteocalcin (OCN; a late marker ofosteoblast maturation), are also shown (FIG. 1 A, B).

Because RC cell cultures comprise a heterogeneous mixture of cell typesand osteoblasts at different differentiation stages, we sought next toconfirm that ERRα is expressed by osteoblast lineage cells. To do this,we used globally-amplified (poly(A) PCR) cDNA pools prepared previouslyfrom single isolated osteoblast colonies at different stages ofdifferentiation (Liu et al., 1994; Liu and Aubin, submitted). Coloniesused were selected based on their molecular phenotypes (relativeexpression levels of collagen type I (COLL I), OPN, bone sialoprotein(BSP), ALP and OCN). ERRα was amplified in each cDNA pool with specificprimers for sequences in the 3′ UTR of ERRA and found to be expressed atall developmental times. Notably, however, levels were generally lowerin more primitive progenitors (A) and osteoblastic cells associated withmineralized nodules (E), and higher in more mature precursors (B),preosteoblasts (C) and osteoblasts (D) (FIG. 2).

Immunocytochemistry was performed to determine whether ERRα protein isexpressed in RC cell cultures. A Western blot of HeLa cell extracts wasused to confirm the specificity of the ERRα antiserum. A singleimmunoreactive band was detected at 53Kd (FIG. 3A). In extracts of thecontrol osteoblastic cell line, MC3T3-E1, ERRα was almost undetectable,however, a strong and single band at 53kd was observed in cell extractsof the same line transfected with an ERRα expression vector (FIG. 3B).

ERRα protein was found widely distributed in most, if not all, cells inRC cell cultures at all times analysed, including early proliferationstages (FIG. 3C), confluence (FIG. 3D), when nascent nodules wereforming (FIG. 3E) and when nodules were mineralizing (FIG. 3F, G). Noteespecially, however, that staining for ERRα was more intense in theosteoblasts associated with both early and late bone nodules than in thesurrounding fibroblastic cells (FIG. 3E, F). Interestingly, while ERRαis primarily found in the cytoplasm and perinuclear location at days 2(FIG. 3C) and 4 (data not shown), by day 6 and thereafter, including inmature osteoblasts, nuclear label is prominent (FIG. 3 E-G).

For comparison, protein expression of four osteoblast markers, ALP (FIG.31), BSP (FIG. 3J), OCN (FIG. 3K) and OPN (FIG. 3L), is also shown. Aspredicted, ERRα is co-expressed in osteoblasts with OPN, which wasdescribed earlier as a target gene of ERRα by cotransfection studies(Bonnelye et al 1997; Vanacker et al, 1998), supporting the hypothesisthat ERRα may regulate OPN in osteoblasts in vivo as well. Clearly, ERRαis also highly co-expressed in cells with ALP, OCN and BSP.

Example 2

ERRα is Also Expressed in Osteoblastic Cells In Vivo in Fetal andPostnatal Rat Calvaria

To extend the observations made in vitro to bones in vivo,immunocytochemistry was performed on sections of 21 d. fetal ratcalvaria, the same bones used for preparation of cell cultures.Consistent with the in vitro results, ERRα was found in all detectablecohorts of osteoblasts from those associated with nascent bone at theosteogenic front (FIG. 4 A) to those in the more mature growing bonetrabecula (FIG. 4B) and remodeling bone (FIG. 4C). Consistent with theRT-PCR results on single bone nodules (cf. FIG. 2), ERRS was alsodetectable in sutural cells (arrows, FIG. 4A), preosteoblasts,osteoblasts (FIG. 4B, C) and osteocytes (FIG. 4B). ERRα was also high inthe osteocytes present in postnatal (4 week) rat calvaria, suggestingthat ERRα may be involved not only in the formation but also in themaintenance of bone. Also consistent with the in vitro cell labeling,ERRα in fetal calvaria in vivo is co-expressed in cells with ALP, OPN(FIG. 4 F, H), BSP (FIG. 4G) and OCN (FIG. 4I).

Example 3

Inhibition of ERRα Expression Blocks the Proliferation of RC Cells andTheir Differentiation to Mature Bone-forming Osteoblasts

Antisense oligonucleotides form DNA:RNA duplexes with specific mRNAspecies, thereby blocking binding of the mRNA to the 40S ribosomalsubunit and preventing translation (Reddy et al., 1994). Preliminaryexperiments were done to determine effective oligonucleotideconcentrations that were not toxic (not shown) and the specificity ofthe antisense was also confirmed by immunocytochemistry on bone nodules.After 24 h of treatment or not with sense or antisense oligonucleotides,ERRα was detectable in bone nodules in untreated cultures and thosetreated with 1 μM sense oligonucleotides but was almost undetectable inbone nodules present in cultures treated with 1 μM antisense (data notshown).

To dissect the possible involvement of ERRα in osteoblastdifferentiation and bone formation, RC cells were treated at differentdevelopmental times from early proliferation stages until mineralizednodule formation.

When RC cells were treated between days 1-6, the proliferation stage, asignificant and specific dose-dependent decrease in cell number (30% at1 μM and 40% at 2 μM), was found at day 6 in dishes treated withantisense compared to sense or untreated controls (FIG. 5A). Theseresults suggest that ERRα may play a role in the proliferation or veryearly differentiation phases of RC cells. To analyze the underlyingmechanism of ERRα action during the proliferation phase, expression ofearly markers of osteoblast differentiation (ALP, BSP, OPN, cbfa1,COLLI), proliferation (Cyclin D1, c-Fos) and apoptosis (Bcl2, Bax) wereassessed at day 6 (FIG. 5B). BSP and cbfal were reduced significantly(FIG. 5C, D); Cyclin D1 was also reduced but it did not reachstatistical significance. On the other hand, ALP, OPN, COLLI, c-Fos, Baxand Bcl2 were not significantly affected (FIG. 5C, D and data notshown). To determine if treatment during the proliferation time windowcaused a sustained alteration in differentiation, terminaldifferentiation/bone nodule formation was assessed at day 15 in culturestreated between days 1-6 with antisense and then switched to normaldifferentiation medium. A significant decrease in mineralized bonenodule number, i.e., 29% at 1 μM and 45% at 2 μM antisenseoligonucleotides (FIG. 6A), was seen. Concomitantly, ALP and BSPexpression remained lower than levels seen in control or sense-treatedcultures, while OPN, OCN and COLLI were not significantly altered (FIG.6B, C).

To determine whether ERRα also plays a role in osteoblastdifferentiation independently of an effect on proliferation, RC cellswere treated with the antisense oligonucleotide beginning at day 5(after cells had reached confluence and proliferation was decreased) today 11. Although cell number was decreased slightly by day 15 (19% at 1μM and 35% at 2 μM) in antisense-treated cultures, a strikingdose-dependent decrease in mineralized bone nodule formation was seen,i.e., 30% at 0.5 μM, 60% at 1 μM and 100% decrease at 2 μM; the senseoligonucleotides had a small non-specific non-dose-dependent effect onnodule numbers. A similar inhibition of bone nodule formation was alsoobserved when the osteoblastic cell line MC3T3-E1 was treated with theantisense oligonucleotides over the comparable time period. Inantisense-treated cultures, ALP-positive colonies were present and largein diameter but flat, suggesting that inhibition of ERRα blockeddifferentiation at an early stage such that progression to matrixdeposition and maturation was reduced. Consistent with thisinterpretation, Cbfa1, BSP and OCN were all decreased inantisense-treated cultures whereas OPN, COLLI and ALP were not affected.Immunocytochemistry confirmed the decrease in OCN- and BSP-expressingcells, but the maintenance of ALP expression in incipient bone nodules(data not shown).

To determine whether ERRα also plays a role at later stages, RC cellswere treated between days 9-15, when differentiation is well-progressedand nascent nodules are becoming three-dimensional. At this stage also,while sense oligonucleotides had a small non-specific, non-dosedependent effect on nodule number, antisense oligonucleotides caused asmall but nevertheless dose-dependent and significant decrease in thenumber of mineralized bone nodules formed, i.e., 17% at 1 μM and 27% at2 μM. In parallel, OCN and OPN but not BSP, COLLI or ALP were decreased.Interestingly, those bone nodules that did form in antisense-treatedcultures appeared to cover a greater surface area compared to those incontrol cultures (data not shown).

Example 4

Overexpression of ERRα Increases Differentiation and Bone NoduleFormation in RC Cells

ERRα expression was upregulated by transient transfection of RC cells atday 5 when cells were between 50-60% confluent. By using a CMV-βGalcontrol vector, it was estimated that a maximal efficiency oftransfection of 10-15% was obtained, which resulted in a 30% increase inERRα levels observed on a Northern blot (FIG. 7A). At day 15, asignificant increase (15%) in number of mineralized bone nodules wasobserved (FIG. 7B). In parallel, we assessed expression levels ofosteoblast markers (ALP, OPN, BSP, COLLI, OCN) 72 h after transfection,at day 10 when bone nodules had started to form and at day 15 whennodules are formed and mineralized. An increase in OPN was observed at72 h and d 10 in cultures overexpressing ERRα, consistent with previousdata describing OPN as a target gene of ERRα in reporter assays(Bonnelye et al, 1997a, Vanacker et al, 1998) (FIG. 7C). COLL I was alsoincreased at the early time points, suggesting a role for ERRα in theformation of a major component of the bone extracellular matrix. OCN, amature osteoblast marker expressed only at later differentiation times,was undetectable at 72 h, but was increased at both day 10 and 15,whereas BSP was increased 72 h after transfection but not detectablyaltered at later times. Finally, ALP, which was increased 72 h aftertransfection, was lower at days 10 and 15 (FIG. 7C).

Example 5

ERRα mRNA is More Highly Expressed in RC Cell Cultures Than Either ERαand ERA mRNA and Expression Patterns Vary

The expression of ERRα mRNA in RC cultures and single bone nodulesprompted a comparison of the levels of its expression with those of thetwo estrogen receptors, ERα and ERA When RT-PCR was done with primersspecific for each of these three receptors, ERRα was found to beexpressed at significantly higher levels than either ERα and ERβ and thetwo estrogen receptors were themselves present at different levels(i.e., ERRα was easily detected at 25 cycles, while 35 cycles and 40cycles were required to detect ERα and ERβ respectively). In addition,the expression patterns of the three receptors over theproliferation-differentiation time course in RC cell cultures wasstrikingly different. Similarly to ERRα, which decreased slightly, ERAdecreased markedly over time in −Dex cultures, whereas ERα increased(FIG. 8A, B). On the other hand, both ERRα and ERβ decreased over timein +Dex cultures, but ERα did not (FIG. 8A, B).

Example 6

ERRα, ERα and ERβ Proteins are Also Expressed in RC Cultures, But OnlyERRα and ERα are Detectable in Bone Nodules In Vitro

To compare the in vitro localization of ERRα with the ER's in RCcultures, immunocytochemistry was performed with polyclonal antibodiesspecific for ERα and ERβ (Santa Cruz, Calif.; Shim et al, 1999; Saji etal, 2000; Tremblay et al, 1999) and for ERRα.

Interestingly, ERα was detected in RC cells at all times analysed fromearly proliferation stages through nodule formation and mineralizationwith especially strong labeling of osteoblastic cells in nodules (datanot shown). ERβ, on the other hand, was more difficult to detect at anytime other than in early proliferating cultures; in particular, ERβ wasseldom detected in osteoblastic cells in bone nodules (data not shown).

ERR protein was found more widely distributed in RC cell cultures thaneither ERα or ERβ. ERRα was found in most if not all cells in RC cellcultures from early proliferation stages through mineralized noduleformation. Interestingly, ERRα was found localized in the nucleus as wasERα in mature osteoblasts. ERcx was mainly nuclear from day 2 to day 6but thereafter was cytoplasmic and nuclear, while ERβ was primarilyperinuclear in all cells in which it could be detected (data not shown).

Example 7

ERRα is More Highly and Widely Expressed In Vivo in Fetal Rat Calvariathan ERα and ERβ

To extend the observations made in vitro to bones in vivo,immunocytochemistry was performed on 21 d. fetal rat calvaria sections(data not shown). Consistent with the in vitro results, strikinglydifferent expression patterns were seen for ERRα, ERα and ERβ. ERRα wasfound in all detectable cohorts of osteoblasts from those associatedwith nascent bone at the osteogenic front to those in more mature bonetrabeculae including remodeling bone. ERα, on the other hand, was notdetected in any cells in the suture or osteogenic front, but wasdetected in some osteoblasts associated with more mature and remodelingbone. ERβ was detected in a pattern virtually reciprocal to that of ERα,i.e., it was present in sutural cells and cells at the osteogenic front,but it was virtually undetectable in osteoblastic cells in more matureand remodeling bone, which is consistent with the mRNA expression in RCcells and the expression in bone nodules. Based on staining intensity,and in keeping with the RT-PCR results, ERRα was more highly expressedthan either ERα and ERβ in vivo.

Example 8

ERRα mRNA is Also Expressed Throughout All Osteoblast Proliferation andDifferentiation Stages in Bone Marrow Stromal Cell Cultures In Vitro

The calvaria bone, an example of a flat bone, forms by a process ofintramembranous ossification, while many other bones form by a processof endochondral ossification. An example of a bone forming byendochondral ossification is the femur, and osteoprogenitor cells alsoreside in the bone marrow stroma of the femur. ERRα mRNA expressionlevels were therefore also assessed over a proliferation-differentiationtime course by Northern blotting of primary rat bone marrow (RBM) cellpopulations grown in the presence (+Dex) or absence (−Dex) ofdexamethasone (Dex), a stimulator of differentiation in this model as itis in the calvaria-derived cells (data not shown). Under both growthconditions, ERRα mRNA was expressed at all times assessed, includingproliferation (day 4), early nodule formation (day 6-9) and nodulemineralization (day 14-17) phases. There was a trend towards increasedERRα expression at early and very late stages, the latter being whenmineralized nodules are present, but otherwise levels were relativelyconstant.

Example 9

ERRα mRNA is More Highly Expressed in RBM Cell Cultures Than Either ERαand ERD mRNA and Expression Patterns Vary

The differential expression of ERRα ERα and ERβ mRNA in RC cultures andsingle bone nodules prompted a comparison of the levels of the threereceptors in RBM cultures as well. When RT-PCR was done with primersspecific for each of the three receptors. As in the RC model, ERRα wasexpressed at significantly higher levels than either ERα and ERβ and thetwo estrogen receptors were themselves present at different levels(i.e., ERRα was easily detected at 25 cycles, while 35 cycles and 40cycles were required to detect ERα and ERβ respectively) in the RBMmodel (FIG. 9A,B). In addition, the expression patterns of the threereceptors over the proliferation-differentiation time course in RBM cellcultures was strikingly different. Whereas ERRα mRNA expresson wasrelatively uniform over the time course analysed, with or without Dex inthe medium, ERα increased late in the proliferation phase (day 4-9) anddecreased thereafter, although levels remained higher than at earliesttimes in −Dex cultures. ERα also increased during the proliferationphase, but peaked later than ERα, i.e., at day 11 (early 30differentiation phase) and then also decreased. Dex appeared to have asmall inhibitory effect on the peak levels reached by both ERα and ERβ.

Example 10

ERRα is Also Expressed in Osteoblastic Cells in Fetal and Postnatal RatFemur

To extend the observations made in vitro to bones in vivo,immunocytochemistry was performed on sections of 21 d. fetal and adultrat femurs (data not shown). Consistent with the in vitro results, ERRαwas highly expressed in osteoblasts associated with the growingtrabecular and cortical bone. ERRα was also found in the osteocytespresent in cortical bone, and in the osteocytes present in the secondaryossification zone, trabecular bone and cortical bone in postnatal (24days) rat femur; ERRα is also highly expressed in the bone marrow ofthese animals. These data suggest that ERRα may be involved not only inthe formation but also in the maintenance of the bone of the axialskeleton.

Example 11

ERRα Expression is Stimulated By Estrogen, Vitamin D3, and TGFβ andInhibited by PTH-1-34 in Proliferating RC Cells Cultures.

To determine whether other hormones or growth factors that influenceproliferation and differentiation in RC cell cultures may modulate ERRαlevels, we treated RC cells continuously with estrogen (17β-estradiol,E2; 10⁻⁸M), 1,25(OH)₂ vitamin D₃ (10⁻⁹M), PGE₂ (10⁻⁹ M), TGFβ (10⁻¹⁰ M)or PTH-1-34 (10⁻¹¹ M). E2 (40% increase), D3 (47% increase) and TGFβ(400%) stimulated expression of ERRα mRNA at day 6, but not later (FIG.10 A,B). An acute exposure of RC cells to E2, D3 or TGFβ for 24 hoursbeginning at day 9 (nascent nodule formation) or day 15 (mature nodulesDresent) (E2 and D3) or day 7 and day 15 (TGFβ had no effect on ERRαmRNA levels (FIG. 10, A, B). PTH-1-34 inhibited ERRα at day 6 (15%) andday 15 (26%), but increased (20%) it at day 12 (FIG. 10C).Interestingly, an acute exposure to PTH-1-34 at day 7 or day 16decreased ERRα by 25% and 33% respectively (FIG. 10C). Neither PGE₂ norDex had any significant effect on ERRS expression.

Example 12

ERRα Expression is Stimulated By Estrogen in Mouse Femur In Vivo

ERRα expression was analysed in samples of mRNA extracted from totalfemurs or femurs from which bone marrow had been flushed of mice treatedwith 17β-estradiol at doses known to elicit a large anabolic effect onendosteal bone (Samuels et al, 1999). In samples of total femoral mRNA,E2 had a small stimulatory effect on ERRα mRNA levels that was mostevident days 2, 12 and 16 (FIG. 11B). In the mRNA extracted from femursfrom which bone marrow had been removed, a marked stimulatory effect ofE2 on ERRα levels was evident at day 1, 2 and 4 (FIG. 11A, B).

Example 13

ERRα Expression is Stimulated in Ovariectomized Rats

ERRα is expressed in adult osteocytes in calvaria and long bone,suggesting a function for ERRα during adult life and in diseases ofbone, including e.g., those characterized by decreased bone mass such asosteoporosis. Ovariectomized (OVX) rats are a model for humanpostmenopausal (estrogen loss-induced) osteoporosis. Four weeks aftersurgery, ERRα expression was increased in osteocytes in the secondaryossification zone and cortical bone (FIG. 12D,H) of long bone andosteoblasts associated with trabecular bone (FIG. 12H, arrows) in OVXcompared to sham-operated (FIG. 12A, C, E, G) rats. High expression ofERRα was also found in the abundant osteoclasts in OVX rats (FIG. 121,arrows). As indicated above, ERRα is expressed in adult calvaria, a sitethought not to be affected by OVX, and no striking differences werefound in staining intensities in sections from sham versus OVX rats(FIG. 12J, K).

REFERENCES

-   Aubin,J E, and Liu, F. (1996) in Raisz L G, Rodan G A, Bilezikian, J    P (eds) Principles of Bone Biology. Academic Press, San Diego,    pp1-45.-   Bain, S D, Bailey,M C, Celino,D L, Lantry, M M, Edwards, M W. (1993)    J, Bone and Mineral Research, 8:435-442-   Barreback, E. D. (1995) Antibody Engineering, 2d. Edition, Oxford    University Press-   Bellow,C. G., and Aubin,J. E.(1989) Dev. Biol. 133,8-13-   Bellow,C. G., Aubin,J. E., Heersche, J. N. M. and    Antosz, M. E. (1986) Calcified Tissue Int.,38: 143-154.-   Bonnelye E , J M. Vanacker, X. Desbiens, T. Dittmer, A. Begue, J.    Aubin, V. Laudet and B. Fournier. Mol Endocrinol, 11., 1997: 905-916    (a)-   Bonnelye E, J M. Vanacker, S. Airic, N. Spruyt, B. Fournier, X.    Desbien and V. Laudet-   MOD, 65, 1997: 71-85 (b)-   Braidman, J P., Davenport, L K, Howard Carter, D, Selby, P L, Mawer.    E B, and Freemont, A J, (1995), LK.(1995) J, Bone and Mineral    Research, 10:74-80-   Buckley, M F, Sweeny, K J E, Hamilton, J A, Sini, R L, Manning, D L,    Nicholson, R I, DeFazio, A Watts, C K W, Musgrove, E A and    Sutherland, R L. (1993) Oncogene 8:2127-2133.-   Case, A M and Reid, R L (1998) Arch Intern Med 158 (13):1405-12.-   Chirgwin, J. J., Przbyla, A. E., MacDonald, R. J., and    Rutter, W. J. (1979) In Current CP protocols . pp.4.2.1-4,2.8 John    Wiley&Sons, Inc.-   Clarke, R, Dickson, R B, and Lippman, M E. (1992) Crit. Rev. Oncol.    Hematol. 12:1-23.-   Da Silva, J A, Larbre, J P, Seed, M P, Cutolo, M, Villaggio, B,    Scott, D L, Willoughby, D A (1994)-   J Rheumatol 21 (2):330-7-   Denhardt, D. T. and Guo, X. (1993) FASEB J.,7,1475-1482-   Denhardt, D. T and Noda, M (1998) J Cell Biochemistry Supp    30/31:92-102.-   Ducy, P, Zhang, R, Geoffroy, V, Ridall, A L and Karsenty, G. (1997)    cell 89 (5): 747-54.-   Enmark, E and Gustafsson, J-A. (1996) Mol.Endocrinol.,10,1293-1307-   Eriksen, E F, Colvard, D S, Berg, N J, Graham, M L, Mann, K G,    Spelsberg, T C, and Riggs, B L 1988 Science 241:84-86-   Genovese, C, Rowe, D and Kream, B (1984) Biochemistry 23: 6210-6216.-   Giguere V, Yang N. Segui P, Evans R M 1988 Nature 331: 91-94-   Green, S., Walter, P., Kumar, V, Krust, A., Bornert, J. M.,    Argos, P. and Chambon, P. (1986) Nature, 320,134-139.-   Green, S. and Chambon, P (1991) Molecular Mechanisms, Cellular    Functions, Clinical Abnormalities.Malcom G.Parker Ed., Academic    Press, Harcourt Brace Jovanovich, Publishers, London.-   Gronemeyer H, Laudet V 1995 Transcription factors 3: nuclear    receptors. Protein profile. 2: 1173-1308-   Grynpas, M D, Huckell, C B, Reichs, K J, Derousseau, C J, Greenwood,    C, Kessler M J (1993) J, Bone and Mineral Research 8(8): 909-17-   Harris, S A, Tau, K R, Turner, R T and Spelsberg, T C, (1996) in    Raisz L G, Rodan G A, Bilezikian, J P (eds) Principles of Bone    Biology. Academic Press, San Diego, pp507-520.-   Hines, K. L, Christ, M and S. M Wahl. (1993) Immunomethods. 3:13-22.-   Hong, H, Yang, L, and Stallcup, M R (1999) J. Biol. Chem, 274 (32):    22618-22626.-   Horwitz et al., (1999), Nat. Med. 5(3): 309-313.-   Hoyland, J A, Baris, C, Wood, L, Baird, P, Selby, P L, Freemont, A J    and Braidman, I P (1999)-   J Patho 188 (3): 294-303-   Huiskes, R, Ruimerman, R, Van Lenthe, G A and Janssen, J D. (2000)    Nature 405: 704-706.-   Johnston, S. D., X. Liu, F. Zuo, T. L. Eisenbraun, S. R.    Wiley, R. J. Kraus and J. E. Mertz. Mol.Endocrinol. (1997) 11:    342-352.-   Korach, K. S. (1994). Science, 266, 1524-1527.-   Komori, T, Yagi, H, Nomura, S, Yamaguchi, A, Sasaki, K, Deguchi, K,    Shimiz, Y, Bronson, R T, Gao, Y H, Inada, M, Sato, M, Okamoto, R,    Kitamura, Y, Yoshiki, S, and Kishimoto, T. (1997) cell 89(5):755-64.-   Kuiper, G. G. J M,. Enmark, E., Pelto-Huikko, M., Nilsson, S. and    Gustafsson, J. A. (1996) Proc.Natl Acad. Sci. USA, 93, 5925-5930.-   Komm, B S, Teruening, C M, Benz, D J, Graeme, K A, Gallegos, A,    Korc, M,-   Greene, G L, O'Malley B W and Haussler, M R 1988 Science 241:81-84.-   Liu, F., Malaval, L., Gupta, A. K., and Aubin, J. E.(1994) Dev.Biol.    166,220-234.-   Liu, F. and Aubin, J. E. (2000) Submitted.-   Malaval, L., Liu, F., Roche, P., and Aubin, J. E. (1999) J. Cell.    Biochem., 74, 616-627.-   Masi, A T, Freigenbaum, S L and Chatterton, R T (1995) Semin    Arthritis Rheum 25 (1):1-27-   Noda, M., Yoon, K., Thiede, M., Buenaga, R., Weiss, M., Henthorn,    P., Harris, H., and Rodan, G. (1987) J.Bone Miner. Res.2,161-164.-   Onoe, Y, Miyaura, C, Ohta, H, Nozawa, S and Suda, T, (1997)    Endocrinology 138 (10): 4509-4512.-   Pacifici, R. (1996) J.Bone Miner.Res., 11, 1043-1051.-   Pettersson K, Svensson K, Mattsson R, Carlsson B, Ohisson R,    Berkenstam A 1996 Mech. Dev. 54:211-213-   Reddy, S V, Takahashi, S Dallas M, Williams R E, Neckers L and    Roodman G D. (1994) J.Bone Miner.Res., 9, 753-757.-   Rosner, I A, Goldberg, V M and Moskowitz, R M-(1986) Clin Orthop    213:77-83-   Sabbah, M, Courilleau, D, Mester, J and Redeuilh, G. (1999) Proc    Natl Acad Sci 96:11217-11222.-   Saji, S, Jensen, E V, Nilsson, S, Rylander, T, Warner, M and    Gustafsson, J A. (2000) Proc Natl Acad Sci-   97 :337-342-   Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold    Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.-   Samuels, A, Perry, M. J and Tobias, J. H (1999) J, Bone and Mineral    Research, 14:178-186.-   Shigeta, H., Zuo, W., DiAugustine, R., and Teng C, T. (1997) J. of    Molecular Endocrinology. 19, 299-309.-   Shim, W S, DiRenzo, J, DeCaprio, J A, Santen, R J, Brown, M and    Jeng, M H (1999) Proc Natl Acad Sci 96:208-213-   Sladek, R., J. A. Bader and V. Giguere. (1997) Mol.Cell.Biol. 17 :    5400-5409.-   Stein et al. (2000) Cancer 88 (12 Suppl): 2899-2902.-   Tremblay, A, Tremblay, G B, Labrie, F and Giguere, V (1999) Mol Cell    3:513-519.-   Turksen, K., and Aubin, J. E. (1991) J.Cell Biol. 114, 373-384.-   Turksen, K., Bhargava, U., Moe, H. K., and Aubin, J. E. (1992) J.    Histochem. Cytochem.40,1339-1352.-   Turner R T, Riggs B L, Spelsberg T C. (1994). Endocr. Rev. 15:    275-300-   Vanacker, J-M., Pettersson, K., Gustafsson, J-A., and Laudet, V.    (1999). EMBO 18, 4270-4279.-   Vanacker, J.-M., Delmarre, C., Guo, X. and Laudet, V. (1998) Cell    Growth Differ., 9,1007-1014.-   Vega, R B and Kelly, D P. (1997) J. Biol. Chem. 272: 31693-31699.-   Wakeling, A. E, Dukes, M and Bowler, J. (1991) Cancer Res. 51:    3867-3873.-   Weiss, M J, Cole, D E, Ray, K, Whyte, M P, Lafferty, M A, Mulivor, R    A and Harris, H. (1988) Proc Natl Acad Sci 85(20):7666-9.-   Wiley S R, Kraus R J, Zuo F, Murray E E, Loritz K, Mertz J E (1993)    Genes and Dev. 7: 2206-2219-   Windahl, S H, Vidal,O, Andersson, G, Gustafsson, J-A and    Ohisson (1999) J Clin Invest 104:895-901.-   Windahl, S H, Norgard, M, Kuiper, G G J M, Gustafsson, J-A and    Andersson, G, (2000) Bone 26:117-121.-   Xie, W, Hong, H, Yang, N N, Lin, R J, Simon, C M, Stallcup, M R and    Evans, R M. Mol Endocrinol (1999) 13(12);2151-62.-   Yang N, Shigeta H, Shi H, Teng C T (1996) J. Biol. Chem. 271:    5795-5804-   Yang, C and Chen, S (1999) Cancer Res 15; 59 (18): 4519-4524.-   Yang, C, Zhou, D and Chen, S (1998) Cancer Res 58: 5695-5700.-   Zhang, Z and Teng, T. (2000) J. Biol. Chem J 275: 20837-20846.-   Zellar, R. and Rogers, M. (1989). In Current CP protocols .    pp.14.3.1-14.3.14. John Wiley&Sons, Inc.

1. A method for screening a candidate compound for its ability to modulate ERRα activity comprising: (a) providing an assay system for measuring a biological activity of ERRα; and (b) measuring the biological activity of ERRα in the presence or absence of the candidate compound, wherein a change in ERRα activity in the presence of the compound relative to ERRα activity in the absence of the compound indicates an ability to modulate ERRα activity.
 2. The method of claim 1 wherein the change in ERRα activity in the presence of the compound is an increase in activity.
 3. The method of claim 2 wherein the increase in ERRα activity in the presence of the compound indicates its potential efficacy as a promoter of bone formation.
 4. The method of claim 1 wherein the change in ERRα activity in the presence of the compound is a decrease in activity.
 5. The method of claim 4 wherein the decrease in ERRα activity in the presence of the compound indicates its potential efficacy as an inhibitor of bone formation.
 6. The method of claim 3 or 5 wherein the assay system for measuring the biological activity of ERRα comprises a rat calvaria cell culture or a bone marrow stromal cell culture.
 7. The method of claim 1 wherein the ERRα activity is human ERRα activity.
 8. A method for screening a candidate compound for potential ability to modulate ERRα activity comprising screening the compound for its ability to bind to ERRα.
 9. The method of claim 8 wherein the ERRα is human ERRα. 